Avian influenza virus (AIV) and infectious bronchitis virus (IBV) are major respiratory pathogens of poultry, and their co-circulation complicates disease control. Here, we developed a chimeric subunit vaccine, RBD-HA, in which the receptor-binding domain (RBD) of the QX-type IBV spike protein replaced the immunodominant head domain of H9N2 AIV haemagglutinin. Structural analyses showed that RBD-HA formed a stable trimeric assembly, supporting the use of the HA stalk as an antigen-presenting scaffold. In chickens, RBD-HA induced humoral responses against both viruses and protected against homologous and heterologous H9N2 AIV challenge. In parallel, RBD-HA induced IBV-reactive and neutralizing antibody responses and provided protection against QX-type IBV challenge. The vaccine also elicited cross-reactive neutralizing activity and reduced viral shedding and tissue damage after H6N6 AIV challenge. These findings provide proof of concept for a bivalent subunit vaccine targeting two major avian respiratory viruses.

Host long non-coding RNAs (lncRNAs) are emerging as critical regulators of influenza A virus (IAV) pathogenesis. Still, the functional landscape of avian host lncRNAs remains largely unexplored. In this study, we identified a novel H9N2induced transcript lncRNA, lncGVRP1, which serves as a conserved positive regulator of IAV replication across H9N2, H1N1, and H3N2 subtypes. In addition, we discovered that lncGVRP1 acts as a functional lncRNA containing a hidden open reading frame (ORF). This ORF encodes a novel 74-amino acid micropeptide, named GVRP1-ORF. Functional rescue experiments demonstrated that the enhancement of viral activity by the lncGVRP1 is strictly dependent on the peptide it encodes. Overexpression of the GVRP1-ORF recapitulated the pro-viral effect achieved by overexpressing the lncRNA. In contrast, an ORF-deleted mutant entirely failed to promote viral replication. Mechanistically, lncGVRP1 facilitates viral propagation by significantly suppressing the host type I interferon (IFN) response and downstream interferon-stimulated genes (ISGs). Furthermore, transcriptome-wide analysis indicated that lncGVRP1 modulates critical cellular machineries, including FoxO signaling and lysosomal trafficking. Collectively, our findings reveal a unique mechanism in which a lncRNA-derived micro-peptide hijacks host immunity to support viral persistence. Moreover, this micro-peptide may serve as a potential therapeutic target against influenza infection.

Circular RNAs (circRNAs) represent a class of covalently closed non-coding RNA molecules that exert vital regulatory effects on host-pathogen interplay. The H9N2 subtype of avian influenza virus (AIV) is a widespread pathogen on a global scale, inflicting considerable economic damage to the poultry sector and harboring potential risks of cross-species transmission to humans. Despite growing evidence suggesting that non-coding RNAs can modulate the replication of influenza viruses, the expression patterns and functions of avian-derived circRNAs during H9N2 AIV infection have been largely unclear. Here, we conducted a systematic investigation into the expression dynamics of circRNAs in DF1 cells infected with H9N2 AIV by high-throughput RNA sequencing technology. A total of 139 differentially expressed circRNAs were identified, with 58 exhibiting upregulation and 81 showing downregulation relative to non-infected control cells. Of note, a circRNA originating from exon 2 of the insulin receptor (INSR) gene displayed consistent upregulation during viral infection. Functional assays verified its contributing role in the replication of H9N2 AIV. Specifically, siRNA-mediated knockdown of circ-INSR significantly suppressed H9N2 AIV replication. This study is the first to identify circ-INSR as a host factor contributing to influenza virus replication. Our results provide a basis for developing circRNA-based strategies against H9N2 avian influenza virus.

Background: Influenza A virus (IAV) remains a major global health threat, and antiviral resistance underscores the need for innovative host-directed therapies. Circular RNAs (circRNAs) are emerging regulators of viral infection, but their functional roles and therapeutic potential in IAV infection remain poorly understood. Methods: Whole-transcriptome sequencing was performed to identify IAV-regulated circRNAs. The role of circCRK in viral replication was evaluated by gain- and loss-of-function approaches in vitro and by lipid nanoparticle (LNP)-mediated circCRK silencing in IAV-infected BALB/c mice. RNA pull-down, RNA immunoprecipitation, dual-luciferase reporter, rescue assays, and analyses of ERK/MAPK signaling and viral ribonucleoprotein (vRNP) trafficking were performed to elucidate the underlying mechanism. Findings: circCRK was significantly upregulated following IAV infection and promoted viral replication. Mechanistically, circCRK functioned as a competing endogenous RNA by sponging miR-516b-5p, thereby relieving repression of RAS1556, a transcript derived from RASGRP2. Activation of the circCRK/miR-516b-5p/RAS1556 axis enhanced ERK/MAPK signaling and facilitated vRNP nuclear export. Importantly, LNP-mediated circCRK silencing reduced viral burden, alleviated disease severity, and improved survival in infected mice. Interpretation: These findings identify circCRK as a host factor that promotes IAV replication through regulation of the miR-516b-5p/RAS1556 axis and reveal circCRK targeting as a promising host-directed antiviral strategy.

ABSTRACT Influenza A viruses (IAVs) pose an ongoing threat to humans and other species because of their zoonotic potential. Accumulating evidence has demonstrated that certain long non-coding RNAs (lncRNAs) exhibit differential expression during viral infection and modulate diverse facets of viral pathogenesis. As key regulatory RNAs, lncRNAs participate in fundamental physiological processes and disease progression via a wide array of functional interactions with DNA, RNA, and proteins. Here, we identified ckATP1A1-AS1 as an antiviral host lncRNA that is induced by IAV infection. Functional analyses demonstrated that ckATP1A1-AS1 overexpression restricted infection by multiple IAV subtypes, whereas ckATP1A1-AS1 knockdown enhanced viral replication. Mechanistically, during IAV infection, the transcription factor JUN transcriptionally activates ckATP1A1-AS1, which further enhances the expression of interferon-β and key interferon-stimulated genes, thereby positively regulating type I interferon immune responses. Furthermore, ckATP1A1-AS1 interacts directly with the viral nucleoprotein, competitively disrupting its binding to importin α5, impairing its oligomerization, and blocking the nuclear import of viral ribonucleoprotein complexes. Consequently, ckATP1A1-AS1 suppresses viral ribonucleoprotein assembly and reduces viral polymerase activity. These findings establish ckATP1A1-AS1 as a key antiviral lncRNA that restricts IAV replication by coordinating innate immune signaling and directly targeting several steps in the viral replication cycle. IMPORTANCE Accumulating evidence indicates that host long non-coding RNAs (lncRNAs) play important roles in regulating virus–host interactions during influenza A virus (IAV) infection. However, the functions and mechanisms of action of most IAV-associated lncRNAs remain unclear. This study identifies the novel chicken antisense lncRNA ckATP1A1-AS1 as a key antiviral factor with a unique dual mechanism: it is transcriptionally activated by transcription factor JUN and, in turn, upregulates the expression of interferon-β and key interferon-stimulated genes to positively regulate the type I interferon immune response. It directly interacts with viral nucleoprotein, competitively disrupting the binding of nucleoprotein to importin α5 and impairing nucleoprotein oligomerization, thereby suppressing viral ribonucleoprotein assembly and reducing viral polymerase activity.

IntroductionCircular RNAs (circRNAs) are covalently closed endogenous RNAs that regulate gene expression at the post transcriptional level and have been implicated in antiviral immunity. However, their functional roles and regulatory mechanisms during influenza A virus (IAV) infection remain incompletely defined.MethodscircZFYVE1 expression was profiled in A549 cells infected with the WSN strain of IAV using RT qPCR under time course and dose response conditions. The mechanistic function of circZFYVE1 was investigated through miRNA target prediction, luciferase reporter assays, and functional assays assessing its role as a competing endogenous RNA (ceRNA) and its impact on innate antiviral signaling.ResultscircZFYVE1 expression was induced in a time and dose dependent manner following WSN infection. Mechanistically, circZFYVE1 acts as a ceRNA by sponging hsa miR 4435, thereby relieving miRNA mediated suppression of the processing body associated protein LSM14A and enhancing innate antiviral signaling during infection.DiscussionThese findings define a circRNA–miRNA–mRNA regulatory axis that links IAV infection to innate antiviral responses, providing new insights into circRNA mediated host defenses. The circZFYVE1/LSM14A pathway represents a potential target for future studies aimed at modulating endogenous antiviral immunity.

Prior research has indicated that the gut-lung-axis can be influenced by the intestinal microbiota, thereby impacting lung immunity. Rifaximin is a broad-spectrum antibacterial drug that can maintain the homeostasis of intestinal microflora. In this study, we established an influenza A virus (IAV)-infected mice model with or without rifaximin supplementation to investigate whether rifaximin could ameliorate lung injury induced by IAV and explore the molecular mechanism involved. Our results showed that IAV caused significant weight loss and disrupted the structure of the lung and intestine. The analysis results of 16S rRNA and metabolomics indicated a notable reduction in the levels of probiotics Lachnoclostridium, Ruminococcaceae_UCG-013, and tryptophan metabolites in the fecal samples of mice infected with IAV. In contrast, supplementation with 50 mg/kg rifaximin reversed these changes, including promoting the repair of the lung barrier and increasing the abundance of Muribaculum, Papillibacter and tryptophan-related metabolites content in the feces. Additionally, rifaximin treatment increased ILC3 cell numbers, IL-22 level, and the expression of RORγ and STAT-3 protein in the lung. Furthermore, our findings demonstrated that the administration of rifaximin can mitigate damage to the intestinal barrier while enhancing the expression of AHR, IDO-1, and tight junction proteins in the small intestine. Overall, our results provided that rifaximin alleviated the imbalance in gut microbiota homeostasis induced by IAV infection and promoted the production of tryptophan-related metabolites. Tryptophan functions as a signal to facilitate the activation and movement of ILC3 cells from the intestine to the lung through the AHR/STAT3/IL-22 pathway, thereby aiding in the restoration of the barrier. • Rifaximin ameliorated IAV infection-caused lung barrier injury and induced ILC3 cell activation. • Rifaximin alleviated IAV-induced gut dysbiosis and recovered tryptophan metabolism. • Tryptophan mediates rifaximin-induced ILC3 cell activation via the AHR/STAT3/IL-22 pathway.

As the H9N2 subtype avian influenza virus (H9N2 AIV) evolves naturally, mutations in the hemagglutinin (HA) protein still occur, which involves some sites with glycosylations. It is widely established that glycosylation of the H9N2 AIV HA protein has a major impact on the antigenicity and pathogenicity of the virus. However, the biological implications of a particular glycosylation modification site (GMS) have not been well investigated. In this study, we generated viruses with different GMSs based on wild-type (WT) viruses. Antigenicity studies revealed that the presence of viruses with a 200G+/295G- mutation (with glycosylation at position 200 and deletion of glycosylation at position 295 in the HA protein) combined with a single GMS, such as 87G+, 127G+, 148G+, 178G+, or 265G+, could significantly affect the antigenicity of the virus. Pathogenicity assays revealed that the addition of GMS, such as 127G+, 188G+, 148G+, 178G+, or 54G+, decreased the virulence of the virus in mice, except for 87G+. The removal of GMS, such as 280G- or 295G-, increased the pathogenicity of the virus in mice. Further studies on pathogenicity revealed that 87G+/295G- could also enhance the pathogenicity of the virus. Finally, we selected the WT, WT-87G+, WT-295G-, and WT-87G+/295G- strains as our further research targets to investigate the detailed biological properties of the viruses. GMS, which can enhance viral pathogenicity, did not significantly affect replication or viral stability in vitro but significantly promoted the expression of proinflammatory factors to enhance inflammatory responses in mouse lungs. These findings further deepen our understanding of the influence of the glycosylation of the HA protein of H9N2 AIV on the pathogenicity and antigenicity of the virus in mice.

After viral infection, the virus relies on the host cell's complex metabolic and biosynthetic machinery for replication. However, the impact of avian influenza virus (AIV) on metabolites and gene expression in poultry cells remains unclear. To investigate this, we infected chicken embryo fibroblasts DF1 cells with H9N2 AIV at an MOI of 3. Our aim was to explore how H9N2 AIV alters DF1 cells metabolic pathways to facilitate its replication. We employed metabolomics and transcriptomics techniques to analyze changes in metabolite content and gene expression. Metabolomics analysis revealed a significant increase in glutathione-related metabolites, including reduced glutathione (GSH), oxidized glutathione (GSSG) and total glutathione (T-GSH) upon H9N2 AIV infection in DF1 cells. Elisa results confirmed elevated levels of GSH, GSSG, and T-GSH consistent with metabolomics findings, noting a pronounced increase in GSSG compared to GSH. Transcriptomics showed significant alterations in genes involved in glutathione synthesis and metabolism post-H9N2 infection. However, adding the glutathione synthesis inhibitor BSO exogenously significantly promoted H9N2 replication in DF1 cells. This was accompanied by increased mRNA levels of pro-inflammatory cytokines (IL-1β, IFN-γ) and decreased mRNA levels of anti-inflammatory cytokines (TGF-β, IL-13). BSO also reduced catalase (CAT) gene expression and inhibited its activity, leading to higher reactive oxygen species (ROS) and malondialdehyde (MDA) level in DF1 cells. qPCR results indicated decreased mRNA levels of Nrf2, NQO1, and HO-1 with BSO, ultimately increasing oxidative stress in DF1 cells. Therefore, the above results indicated that H9N2 AIV infection in DF1 cells activated the glutathione metabolic pathway to enhance the cell's self-defense mechanism against H9N2 replication. However, when GSH synthesis is inhibited within the cells, it leads to an elevated oxidative stress level, thereby promoting H9N2 replication within the cells through Nrf2/HO-1 pathway. This study provides a theoretical basis for future rational utilization of the glutathione metabolic pathway to prevent viral replication.

As a highly contagious acute respiratory disease, influenza A virus (A/WSN/1933) poses a huge threat to human health and public health. influenza A virus proliferation relies on glucose metabolism in host cells, yet the effects of influenza A virus on glucose metabolism and the underlying molecular mechanisms remain unclear. Here, we created models of WSN virus-infected mice and A549 cells, along with analyzing metabolomics and transcriptomics data, to investigate how WSN virus infection affects host cell glucose metabolism and specific mechanisms. Analysis of metabolites and gene expression showed that WSN virus infection triggers glycolysis in A549 cells, with notable upregulation of hexokinase 2 (HK2), lactate dehydrogenase A (LDHA), hypoxia-inducible factor-1 alpha (HIF-1α), and elevated lactate levels. Additionally, it leads to mitochondrial impairment and heightened reactive oxygen species (ROS) generation. Elevated levels of glucose may enhance the replication of WSN virus, whereas inhibitors of glycolysis can reduce it. Enhancement of HIF-1α activation facilitated replication of WSN virus through stimulation of lactate synthesis, with the primary influence of glycolysis on WSN virus replication being mediated by ROS/HIF-1α signaling. Mice given HIF-1α inhibitor PTX-478 or glycolysis inhibitor 2-Deoxyglucose (2-DG) exhibited reduced lactate levels and decreased WSN virus replication, along with mitigated weight loss and lung damage. In summary, WSN virus-induced glycolysis has been demonstrated to enhance virus replication through the activation of the ROS/HIF-1α pathway, suggesting potential new targets for combating the virus.

IntroductionIntroduction: The influenza virus primarily targets the respiratory tract, yet both the respiratory and intestinal systems suffer damage during infection. The connection between lung and intestinal damage remains unclear.MethodsOur experiment employs 16S rRNA technology and Liquid Chromatography-Mass Spectrometry (LC-MS) to detect the impact of influenza virus infection on the fecal content and metabolites in mice. Additionally, it investigates the effect of influenza virus infection on intestinal damage and its underlying mechanisms through HE staining, Western blot, Q-PCR, and flow cytometry.ResultsOur study found that influenza virus infection caused significant damage to both the lungs and intestines, with the virus detected exclusively in the lungs. Antibiotic treatment worsened the severity of lung and intestinal damage. Moreover, mRNA levels of Toll-like receptor 7 (TLR7) and Interferon-b (IFN-b) significantly increased in the lungs post-infection. Analysis of intestinal microbiota revealed notable shifts in composition after influenza infection, including increased Enterobacteriaceae and decreased Lactobacillaceae. Conversely, antibiotic treatment reduced microbial diversity, notably affecting Firmicutes, Proteobacteria, and Bacteroidetes. Metabolomics showed altered amino acid metabolism pathways due to influenza infection and antibiotics. Abnormal expression of indoleamine 2,3-dioxygenase 1 (IDO1) in the colon disrupted the balance between helper T17 cells (Th17) and regulatory T cells (Treg cells) in the intestine. Mice infected with the influenza virus and supplemented with tryptophan and Lactobacillus showed reduced lung and intestinal damage, decreased Enterobacteriaceae levels in the intestine, and decreased IDO1 activity.DiscussionOverall, influenza infection caused damage to lung and intestinal tissues, disrupted intestinal microbiota and metabolites, and affected Th17/Treg balance. Antibiotic treatment exacerbated these effects. Supplementation with tryptophan and Lactobacillus improved lung and intestinal health, highlighting a new understanding of the lung-intestine connection in influenza-induced intestinal disease.

为探究H9N2亚型禽流感病毒(AIV)和大肠杆菌共感染对无特定病原体(SPF)鸡肺组织和免疫系统的损伤及细胞自噬在共感染致病过程中的作用,选用60只3周龄SPF鸡,随机平均分为对照组、单独细菌感染组、单独病毒感染组、先病毒后细菌共感染组和先细菌后病毒共感染组5组.于处理后第1、3、5和7天各组随机选择3只鸡采集血清和肺组织,利用荧光定量PCR和HE组织学染色技术探究共感染对SPF鸡的肺组织系数、肺组织病理变化和肺组织屏障功能的影响.利用细胞培养、荧光定量PCR、酶联免疫吸附和Western blot等技术探究共感染与细胞自噬的关系以及自噬对共感染炎症反应和辅助性T细胞17(Th17)免疫反应的影响.结果显示:先病毒后细菌共感染试验组导致鸡肺组织病理损伤严重;共感染诱导强烈的细胞自噬,先病毒后细菌组自噬相关因子(LC3、Beclin-1)的蛋白表达水平与其他组相比极显著升高(P＜0.01);同时与其他组相比,先病毒后细菌组炎症因子基因表达水平极显著升高(P＜0.01),Th17免疫相关因子基因表达水平极显著降低(P＜0.01).提示:自噬通过促进共感染中炎性因子释放且抑制Th17免疫反应,造成鸡肺组织严重损伤.本试验为研究病毒与细菌共感染机制提供了科学依据.

Gut-lung axis injury is a common finding in patients with respiratory diseases as well as in animal model of influenza virus infection. Influenza virus damages the intestinal microecology while affecting the lungs. Rifaximin, a non-absorbable derivative of rifamycin, is an effective antibiotic that acts by inhibiting bacterial RNA synthesis. This study aimed to determine whether rifaximin-perturbation of the intestinal microbiome leads to protective effects against influenza infection, via the gut-lung axis. Our results showed that influenza virus infection caused inflammation of and damage to the lungs. The expression of tight junction proteins in the lung and colon of H1N1 infected mice decreased significantly, attesting that the barrier structure of the lung and colon was damaged. Due to this perturbation in the gut-lung axis, the intestinal microbiota became imbalanced as Escherichia coli bacteria replicated opportunistically, causing intestinal injury. When influenza infection was treated with rifamixin, qPCR results from the gut showed significant increases in Lactobacillus and Bifidobacterium populations, while Escherichia coli populations markedly decreased. Furthermore, pathology sections and western blotting results illustrated that rifaximin treatment strengthened the physical barriers of the lung-gut axis through increased expression of tight junction protein in the colon and lungs. These results indicated that rifaximin ameliorated lung and intestine injury induced by influenza virus infection. The mechanisms identified were the regulation of gut flora balance and intestinal and lung permeability, which might be related to the regulation of the gut-lung axis. Rifaximin might be useful as a co-treatment drug for the prevention of influenza virus infection.

Aging is a complex physiological process associated with degenerative disorder of metabolism and immune function, which contributes to the occurrence of senile diseases. The gut microbiota affects systemic inflammation in aging processes probably through metabolism, but their relationship is still unclear. In this study, 16S-rRNA-sequencing technology, gas chromatography-time-of-flight mass spectrometry (GC-TOFMS)–based metabolic profiling, and immune factor analysis combined with advanced differential and association analysis were employed to investigate the correlation between the microbiome, metabolome, and immune factors in male Wistar rats across lifespan. Our findings showed significant changes in the ileum microbiome and serum metabolome compositions across aging process. A two-level strategy was applied to demonstrate that key metabolites associated with age such as 4-hydroxyproline, proline, and lysine were clustered together and positively correlated with beneficial microbes including Bifidobacterium , Lactobacillus , and Akkermansia . Function analysis explored association between serum metabolite class and specific gut bacteria’s metabolism pathways. Further correlation analysis on all the alteration patterns provided an interaction network of main immune factors such as IL-10, IgA, IgM, and IgG with key gut bacteria and serum metabolites. This study offers new insights into the relationship between immune factors, serum metabolome, and the gut microbiome.

In the past few decades, cardiac regeneration has been the central target for restoring the injured heart. In mammals, cardiomyocytes are terminally differentiated and rarely divide during adulthood. Embryonic and fetal cardiomyocytes undergo robust proliferation to form mature heart chambers in order to accommodate the increased workload of a systemic circulation. In contrast, postnatal cardiomyocytes stop dividing and initiate hypertrophic growth by increasing the size of the cardiomyocyte when exposed to increased workload. Extracellular and intracellular signaling pathways control embryonic cardiomyocyte proliferation and postnatal cardiac hypertrophy. Harnessing these pathways could be the future focus for stimulating endogenous cardiac regeneration in response to various pathological stressors. Meanwhile, patient-specific cardiomyocytes derived from autologous induced pluripotent stem cells (iPSCs) could become the major exogenous sources for replenishing the damaged myocardium. Human iPSC-derived cardiomyocytes (iPSC-CMs) are relatively immature and have the potential to increase the population of cells that advance to physiological hypertrophy in the presence of extracellular stimuli. In this review, we discuss how cardiac proliferation and maturation are regulated during embryonic development and postnatal growth, and explore how patient iPSC-CMs could serve as the future seed cells for cardiac cell replacement therapy.

Emerging influenza D viruses (IDVs), the newest member in the genus Orthomyxovirus family, which can infect and transmit in multiple mammalian species as its relatives the influenza A viruses (IAVs). Additional studies of biological characteristics of IDVs are needed; here, we studied the characteristics of IDV nonstructural protein 2 (NS2), which shares the lowest homology to known influenza proteins. First, we generated reassortant viruses via reverse genetics to analyze the segment compatibility and gene interchangeability between IAVs and IDVs. Next, we investigated the locations and exact sequences of nuclear export signals (NESs) of the IDV NS2 protein. Surprisingly, three separate NES regions were found to contribute to the nuclear export of an eGFP fusion protein. Alanine scanning mutagenesis identified critical amino acid residues within each NES, and co-immunoprecipitation experiments demonstrated that their nuclear export activities depend on the CRM1-mediated pathway, particularly for the third NES (136-146aa) of IDV NS2. Interestingly, the third NES was important for the interaction of NS2 protein with CRM1. The findings in this study contribute to the understanding of IDV NS2 protein’s role during nucleocytoplasmic transport of influenza viral ribonucleoprotein complexes (vRNPs) and will also facilitate the development of novel anti-influenza drugs targeting nuclear export signals of IDV NS2 protein.

流感病毒作为一种常见的呼吸道病毒,是人类健康和世界经济的巨大威胁.目前流感治疗遇到病毒高度突变的问题,不断完善已有的控制流感感染方法的同时,也需要开阔视野从宿主反应的角度探索控制流感的新措施,本研究从肠道微生物的角度探索流感造成肺损伤的机制.提前3周将组合抗生素和益生元添加到小鼠饮水中构建不同的肠道微生物环境,小鼠感染流感病毒后运用16S rDNA技术测定结肠微生物组成,蛋白免疫印迹法测定肺部Th17和Treg细胞转录因子RORγT和Foxp3的表达情况,苏木精-伊红染色、荧光定量PCR测定肺损伤状况.结果 表明,提前使用组合抗生素和益生元能够改变肠道菌群组成,通过降低肠道菌群数目和增加肠道拟杆菌相对丰度保护肠道,从而反作用于肺部诱导肺Treg细胞分化,抑制Th17细胞分化,改善流感造成的肺损伤.

[目的]本文旨在研究流感感染小鼠使用抗生素后肠道微生物、代谢物及肠道紧密连接蛋白的变化.[方法]30只雄性BALB/c小鼠随机分为3组:对照组、感染组和抗生素组.感染组和抗生素组同时感染流感病毒,抗生素组感染1 d后饮水中添加组合抗生素,3组小鼠感染9 d后取样.16S rRNA基因测序后进行微生物组学分析;用气相色谱-飞行时间质谱(GC-TOF/MS)法进行代谢组学分析;用免疫组化法和免疫印迹法进行结肠紧密连接蛋白的分析.[结果]与对照组相比,感染组微生物多样性指数(Chao1、Shannon、Simpson)、细菌丰度和代谢物主成分分析(PCA)均无显著差异.与感染组相比,抗生素组微生物多样性指数均极显著下降(P<0.01).拟杆菌门和变形菌门相对丰度极显著增加(P<0.01),厚壁菌门相对丰度极显著降低(P<0.001).抗生素组与对照组在PCA中完全分离,有机酸代谢与筛选出的差异微生物具有较强相关性.感染组和抗生素组的紧密连接蛋白(Claudin-1、Occludin)的表达量均极显著降低(P<0.01),抗生素组Claudin-1的表达量与感染组相比极显著降低(P<0.001).[结论]抗生素降低肠道微生物的多样性,增加致病菌的侵入,减少有益菌的繁殖,加剧流感诱导的肠道菌群失调,减少有机酸的产生,导致更严重的肠道屏障功能损伤.

Evidence has shown that neuromedin S (NMS) and its receptor (NMU2R) are expressed in the hypothalamus, pituitary, and testis of pigs. To determine the potential mechanisms of NMS, we systematically investigated the direct effects of NMS on the hypothalamic-pituitary-testicular (HPT) axis of male pigs in vitro. We initially confirmed that NMU2R distributed in isolated hypothalamic cells, anterior pituitary cells and Leydig cells using immunocytochemistry. Subsequently we investigated the direct effects of NMS on hormone secretion from cells (anterior pituitary cells and Leydig cells) treated with different doses of NMS. The results showed that NMS increase the release of LH and FSH from anterior pituitary cells and testosterone from Leydig cells. NMS up-regulated the expression of NMU2R and GnRH mRNAs in hypothalamic cells, NMU2R, LH and FSH mRNAs in anterior pituitary cells, and NMU2R, STAR, P450 and 3β-HSD mRNAs and the expression of PCNA and Cyclin B1 protein in Leydig cells; moreover, it down-regulated the expression of GnIH mRNA in hypothalamic cells. Using immunofluorescence staining and confocal microscopy, we also demonstrated the colocalization of NMU2R and AR or GnIH in Leydig cells. These data in vitro indicated that NMS may regulate the release and/or synthesis of LH, FSH and testosterone at different levels of the reproductive axis through NMU2R, which provided novel evidence of the potential roles of NMS in regulation of pig reproduction.

Neuropeptide B (NPB) is an endogenous ligand for the orphan G protein-coupled receptors NPBWRI (GPR7) and NPBWR2 (GPR8). Some reports have investigated the role of NPB in the regulation of feeding, energy metabolism and hormone secretion in many species. However, few papers reported the physiological function of NPB in the pig. In this study, we cloned and sequenced the NPB mRNA from a pig, which was found to consist of 123 bases. NPB mRNA expression was detected in central and peripheral tissues by the quantitative fluorescence method. The results showed that NPB mRNA expression was higher in hippocampus, cerebellum, spinal cord, thymus, tonsil, duodenum, cecum, colon, ovary and testis. The distribution of NPB suggested that it may be involved in the regulation of reproductive functions in the pig. Subsequently, the expression and distribution of NPBWR1 and NPBWR2 were found in Leydig cells and ovarian granular cells. We then investigated the direct effect of NPB on pig reproductive cells in vitro. The results showed that different concentrations of NPB (10(-12), 10(-10), 10(-8) and 10(-6) M) promoted the secretion of testosterone in Leydig cells in concentration-dependent manner. Different doses of NPB could promote the secretion of progesterone in ovarian granulosa cells in dose-dependent manner. Low concentrations of NPB (10(-8) and 10(-10) M) promoted estradiol secretion, but high concentrations of NPB (10(-6) M) inhibited its secretion. All the results suggested that the NPB/NPBWR1 or NPBWR2 system may play a role in modulating the reproductive activity in the pig. (C) 2018 Elsevier Inc. All rights reserved.